This application is generally in the area of solid phase extraction, more specifically, in the area of cartridges for use in solid phase extraction.
Solid phase extraction uses analyte/sorbent interactions similar to those used in high performance liquid chromatography (HPLC). Solid phase extraction uses cartridges packed with a variety of sorbents, for example, surface-modified bonded silica sorbents, which selectively retain specific classes of chemical compounds from a group of compounds added to the cartridge. For example, strong cation exchangers can be used to retain cationic drugs, such as amphetamine, from urine samples. Some interactions between sorbent and analyte are relatively strong, for example, sorbents bonded to biotin strongly interact with streptavidin in an analyte sample.
Bonded silica sorbents are often used for chromatographic isolation, in part because a number of different functional groups can be readily bonded to the silica surface. Bonded silicas are also advantageous in that they are rigid supports and do not shrink or swell. Further, they have relatively large surface areas and are stable to a wide range of solvent conditions.
Solid phase extraction is commonly used to clean-up and concentrate compounds in analyte mixtures, and also to exchange solvent systems, for example, from aqueous to organic, at some point prior to a further analysis. Solid phase extraction can be used to achieve these goals.
Solid phase extraction consists of four basic steps, namely, conditioning, retention, rinsing and elution. Conditioning involves preparing a cartridge for reproducible interaction with a sample by solvating the sorbent bed. This is typically done by passing a volume of an appropriate solvent through the cartridge, and then passing through a volume of a solvent system similar to that used in the sample. For example, when conditioning a cartridge for use with aqueous samples, a volume of an alcohol such as methanol might be passed through the cartridge, followed by a volume of water. Retention involves applying the sample to the conditioned cartridge and allowing the compound of interest to be retained on the sorbent surface due to one or more specific chemical interactions. The interactions might be linkage of biotin to streptavidin, or Van der Waals interactions between the hydrocarbon chain of a compound of interest and a hydrocarbon chain on a C18 bonded phase. Components of the sample that bind with less affinity typically pass through the cartridge, resulting in purification of the sample. Rinsing involves passing solvents through the cartridge to rinse away additional compounds other than the compound of interest while leaving the compound of interest adhered to the sorbent bed. Water is commonly used as a rinse solvent for non-polar extractions performed on a C18 sorbent. Elution involves passing an appropriate solvent through the cartridge while it disrupts the interaction between the compound of interest and the sorbent, which allows the compound of interest to be selectively eluted from the cartridge. For non-polar extractions, organic solvents such as alcohols are generally strong enough solvents to disrupt the interaction between most non-polar analytes and a C18 bonded phase.
Automated equipment is often used to perform analytical analyses of a large number of analytes. Solid phase extraction cartridges have been designed which are capable of being used in automated equipment, such as robotic equipment. New sorbents and cartridge systems are constantly being generated for use in detecting the presence of various compounds, for example, environmental contaminants.
Analytical methods have been developed for determining the presence of aryl pyrazole insecticides such as fipronil in agricultural soil and filter paper plaques. However, the previously described methods are limited in that they are not automated, and therefore are limited in their usefulness for evaluating large numbers of soil samples. Moreover, there is a need in the art for analytical methods for determining the amount of pyrazoles in a variety of samples in the agricultural, pharmaceutical and other areas of science.
Thus, there is a continuing need for further analytical methods and detection systems which can be used in automated chemical analyses to detect the presence of potentially hazardous compounds in various environmental samples. The present invention provides such methods and detection systems.
Methods and apparatus are disclosed which are useful for detecting the presence of pesticides, such as pyrazoles including fipronil, in soil, water, blood, tissue, urine and other types of agricultural, medical or pharmaceutical samples. The methods and apparatus of the invention can be used in a wide variety of applications to purify and allow for the quantitation of the amount of pesticide, especially pyrazole, particularly aryl pyrazole, and its metabolites in a given sample.
The present invention provides an apparatus for solid phase chromatography comprising a column having a first opening for receiving a sample and a second opening for discharging an eluate, which column comprises a first separation zone containing amino-functional silica, a second separation zone containing activated carbon, a third separation zone containing magnesium-silica gel or silica gel and a fourth separation zone containing magnesium-silica gel or silica gel, whichever was not used in the third separation zone.
The activated carbon generally is pre-treated with hydrochloric acid prior to use in the apparatus of the invention. The amino-functional silica also generally is treated with organic solvent prior to placement in the apparatus of the invention. In one aspect of the invention, a fifth separation zone is provided containing a drying agent.
In another aspect of the invention, a method for removing aryl pyrazoles from a sample is provided which comprises extracting the aryl pyrazole residues from a sample in a solvent, concentrating the extract to form a concentrated extract, and subjecting the concentrated extract to solid phase chromatography on a column comprising a first separation zone containing amino-functional silica; a second separation zone containing activated carbon, a third separation zone containing magnesium-silica gel or silica gel and a fourth separation zone containing magnesium-silica gel or silica gel, whichever was not used in the third separation zone. The eluate eluting from the column after the chromatography will contain the aryl pyrazole residues and metabolites.
In another aspect of the invention, a method for determining the amount of pesticide residue in a sample is provided, comprising extracting the pesticide residues from a sample in a solvent; concentrating the extract to form a concentrated extract; transferring the concentrated extract to a column comprising a first separation zone containing amino-functional silica, a second separation zone containing activated carbon, a third separation zone containing magnesium-silica gel or silica gel and a fourth separation zone containing magnesium-silica gel or silica gel, whichever was not used in the third separation zone; subjecting the concentrated extract to solid phase extraction by passing at least one organic solvent through the column to obtain an eluate solution; removing the eluate solution from the column; and analyzing the eluate solution to determine the amount of pesticide residue.
In one aspect, the methods and apparatus of the invention may be utilized to isolate a pyrazole and its metabolites from a soil, water or other sample. Where the pyrazole is in a soil sample, the residues of the pyrazole first may be extracted using techniques well known to those of skill in the art. The extracts then are concentrated, and the samples are cleaned up or purified using solid phase extraction, using multiple sorbent cartridges to remove various components other than the compounds of interest (the aryl pyrazoles and their metabolites). The sorbent cartridges include amino-functional silica, activated carbon, magnesium-silica gel and silica gel, or suitable equivalents thereof.
In a preferred embodiment, the column chromatography materials are assembled in a solid phase extraction cartridge. Preferably, the cartridge is a twenty cubic centimeter cartridge. The components for the solid phase extraction can be packed in the cartridge with or without dividing elements between the various components. In one aspect of the invention, the cartridge has frits on top, on the bottom and between the chromatography materials. This facilitates shipping of the cartridges and the use of the cartridges in standard automated analytical equipment, for example, robotic equipment.
Using the methods and apparatus described herein, large numbers of samples can be analyzed in an automated fashion.